Recently I tried to cross a tissue-specific RNAi strain (that possesses rde-1 mutation to make the worm not susceptible to RNAi, but the wild-type
copy of it is present in the form of a construct and it’s expressed under the promoter of a specific tissue) with another mutant. The problem is that
by sequencing I can’t differentiate whether my new mutant has the rde-1 mutation or not, because of the abundance of the wild-type gene. Is there a
way to detect the mutation in the genome? Would be very grateful for any advice!
Construct your strain with an rde-1 balancer so you don’t need to check the allele molecularly. unc-42 sma-1 double works fine to balance rde-1, and I have used it for this purpose before.
Another balancer option you might consider is nT1 [qIs51]; the dominant gfp marker can be really useful in your strain constructions, and you can be a little sloppier about monitoring generations because there’s no real risk of recombination (not that the risk with unc-42 sma-1 is big). Not useful if you’re building with something on LGIV or LGV, but otherwise it’ll save you time and trouble.
Hillel’s right of course. Mine included let-60(n1046) on IV, so I wanted to avoid nT1g. But nT1g is far easier if you don’t have another factor on IV (rde-1 is on V)
Dear all, thank you very much for the useful replies. Dear Snug, I actually didn’t pay attention to the regions that I used to design the primers. Hopefully that will sort out my problem. Thanks
Dear Snug, I actually didn’t pay attention to the regions that I used to design the primers. Hopefully that will sort out my problem. Thanks 